EP3221327A1 - Nukleosidphosphoramidatester und derivate davon, verwendung und synthese davon - Google Patents

Nukleosidphosphoramidatester und derivate davon, verwendung und synthese davon

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Publication number
EP3221327A1
EP3221327A1 EP15805684.6A EP15805684A EP3221327A1 EP 3221327 A1 EP3221327 A1 EP 3221327A1 EP 15805684 A EP15805684 A EP 15805684A EP 3221327 A1 EP3221327 A1 EP 3221327A1
Authority
EP
European Patent Office
Prior art keywords
group
carbon
hydroxyl
alkyl
bonded
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP15805684.6A
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English (en)
French (fr)
Inventor
Mark Stamatios Kokoris
John Tabone
Melud Nabavi
Aaron Jacobs
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Original Assignee
Stratos Genomics Inc
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Publication date
Application filed by Stratos Genomics Inc filed Critical Stratos Genomics Inc
Publication of EP3221327A1 publication Critical patent/EP3221327A1/de
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus
    • C07F9/65746—Esters of oxyacids of phosphorus the molecule containing more than one cyclic phosphorus atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/14—Pyrrolo-pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention is in the field of phosphoroamidate ester compounds, synthetic methods for making said compounds, and methods for the determination of nucleic acids using said compounds, e.g., in the field of single molecule sequencing.
  • nucleotide triphosphates may be synthesized with modifications at the base, sugar, or triphosphate chain. Historically, modification of the triphosphate chain has been mainly used to study enzymatic pathways, which results in hydrolysis and transfer of the phosphate from NTP to another molecule. Modification of sugar and base has served a number of different purposes, from pharmaceutical to diagnostic applications.
  • dNTP deoxyribonucleotide triphosphate
  • 5' phosphoramidates and analogs have increased due to their potential utility in genomic analysis (see, e.g., Shchepinov et al., Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms. Nuc. Acids Res. v. 30(17) pp. 3739-3747 (2002)) and DNA sequencing (see, e.g., U.S Pat. No. 8,324,360 to Kokoris et al.).
  • Certain useful features of 5' phosphoramidate analogs include their ability to exist in a triphosphate form, which can be utilized by many polymerases (see, e.g., Letsinger et al.,
  • Triphosphates are of particular importance as substrates for DNA or RNA polymerase that incorporate the nucleotide analogs into long chain nucleic acids. Generally, triphosphates are synthesized by first preparing the nucleoside monophosphates, which are subsequently converted into
  • NM P monophosphate
  • one technical object forming the basis of the present invention is to provide improved nucleotide phosphoramidate analogs that are further modified on the alpha-phosphate to enable attachment of a variety of application-specific substituents (e.g. tether molecules) and, furthermore, to provide reliable processes for the synthesis of such novel nucleotide analogs.
  • application-specific substituents e.g. tether molecules
  • the present disclosure provides mono and
  • polyphosphoroamidate ester compounds synthetic methods for the preparation of such compounds, compounds useful in the synthetic methods, and uses for the compounds.
  • R 1 is selected from
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R is selected from
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the present disclosure provides synthetic methods and compounds useful therein, for the preparation of mono and polyphosphoroamidate ester compounds.
  • the present disclosure provides a process for forming a phosphoromonoamidate diester 110 comprising contacting compound 100 with compound 105 to provide compound 110, the contacting conducted in the presence of a halide anion source,
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 4 is selected from R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the present disclosure provides a process of forming a phosphate protected N-phosphoroamidate-monoester diphosphate 120 comprising contacting a compound 110 with a compound 115 followed by oxidation to provide com ound 120,
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl; R 4 is selected from
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the present disclosure provides a process of forming a phosphate protected N-phosphoroamidate-monoester triphosphate (125) comprising contacting a compound (120) with a compound (115) to provide, after oxidation, a phosphate protected N-phosphoroamidate-monoester triphosphate (125),
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 4 is selected from
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the present disclosure provides a cyclic phosphite of the formula
  • R 1 is an alkyl group or an oxyalkyi group, either of which is terminally- functionalized, where the terminal functional group is selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl or ester thereof, formyl, hydroxylamino and halogen.
  • the terminal functional group of R 1 may be carbon-carbon double bond; and/or it may be carbon-carbon triple bond; and/or it may be hydroxyl; and/or it may be amine; and/or it may be thiol; and/or it may be carboxyl or ester thereof; and/or it may be formyl; and/or it may be hydroxylamino; and/or it may be halogen.
  • R 1 comprises an alkyl group.
  • R 1 when R 1 is an alkyl group and the functional group is a carbon-carbon triple bond, R 1 may be -(CH 2 ) q - C ⁇ CH where -(CH 2 ) q is the alkyl group, which might also be referred to as an alkylene group, and q is an integer selected from 2-10, e.g., R 1 is 1-hexynyl of the formula - CH 2 CH 2 CH 2 CH 2 C ⁇ CH.
  • R 1 includes an electrophilic group.
  • R 1 includes a nucleophilic group.
  • R 1 includes a carboxylic acid or an ester thereof. I n one embodiment, R 1 is an alkyl group which is terminally-functionalized.
  • R 1 is an oxyalkyl group which is terminally functionalized, where an oxyalkyl group may also be called an oxyalkylene group, and refers to an alkyl group that incorporates one or more oxygen atoms in the form of ether groups.
  • Oxyethylene (-0-CH 2 -CH 2 -) groups and oxypropylene (-0-CH 2 - CH 2 -CH 2 -) groups are exemplary oxyalkyl groups.
  • the oxyalkyl group of R 1 may be formed from one or a plurality of oxyalkyl units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 repeating units.
  • the cyclic phosphite as described herein may be used in a process for forming a N-phosphoroamidate- monoester tri hosphate (160) from the cyclotriphosphite (155) and an azide (105)
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • FIGURE 1 illustrates how a nucleobase triphosphoramidate of the present disclosure may function as a component of a XNTP substrate useful in
  • the present invention is directed to one or more of new compounds, methods for preparing compounds including novel compounds useful in the synthetic methods, and the use of these compounds in, for example, nucleic acid sequencing techniques, each as disclosed herein.
  • nucleic acid sequencing techniques each as disclosed herein.
  • Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms (Ci-C 20 alkyl or Ci -2 o alkyl), and typically from 1 to 12 carbons (Ci-Ci 2 alkyl or Ci_ i2 alkyl) or, in some embodiments, from 1 to 8 carbon atoms (C C 8 alkyl or Ci_ 8 alkyl) or, in some embodiments, from 1 to 4 carbon atoms (Ci-C 4 alkyl or Ci_ 4 alkyl) or, in some embodiments, from 1 to 3 carbon atoms (C1-C3 alkyl or Ci_ 3 alkyl).
  • straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.
  • branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
  • the alkyl group may be substituted or otherwise functionalized with a functional group.
  • substituted alkyl groups can be substituted one or more times with any non-alkyl group, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • the alkyl group may contain one or more carbon-carbon double bonds and one or more carbon-carbon triple bonds along its structure.
  • the term "terminally functionalized alkyl group” and its equivalent term “omega-functionalized alkyl group” refers to an alkyl group that terminates in a functional group.
  • the group -CH2CH2CH2CH2-OH represents a terminally functionalized n-butyl group having hydroxyl as the functional group, where this group may also be described as n-hydroxy C 4 alkyl.
  • Unsubstituted alkyl groups which are optionally functionalized by the presence of carbon-carbon double bonds and/or carbon-carbon triple bonds, are examples of hydrocarbon groups, i.e., groups formed entirely of carbon and hydrogen.
  • the hydrocarbon group is an alkyl group.
  • a C 6 -Ci 6 hydrocarbon has 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms in addition to hydrogen atoms as the only atoms present in the hydrocarbon moiety.
  • each of R 1 and R 13 may be a hydrocarbon alkyl group.
  • each of R 1 and R 13 may be an oxyalkyi group.
  • Oxyalkyi refers to alkyl groups that are separated by oxygen, i.e., alkyl-O- alkyl- etc. and the like.
  • Exemplary alkyl groups in an oxyalkyi unit are ethyl and propyl.
  • oxyalkyi may refer to one or more repeating units of -CH 2 - CH2-O-.
  • -ethyl-O-ethyl-O-ethyl-O-ethyl-O-ethyl is an exemplary oxyalkyi group.
  • the number of repeating alkyl-0 units in an oxyalkyi may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10.
  • Halogen refers to bromide, chloride, iodide and fluoride.
  • Heterocycle and heterocyclyl groups include aromatic and non-aromatic ring compounds (heterocyclic rings) containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, S, or P.
  • heteroatom such as, but not limited to, N, O, S, or P.
  • heterocyclyl groups include 3 to 20 ring members, whereas other such groups have 3 to 15 ring members. At least one ring contains a heteroatom, but every ring in a heteropolycyclic system need not contain a heteroatom.
  • a dioxolanyl ring and a benzdioxolanyl ring system are both heterocyclyl groups within the meaning herein.
  • a heterocyclyl group designated as a C 2 -heterocyclyl can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heterocyclyl can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, and so forth.
  • the number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms.
  • a saturated heterocyclic ring refers to a heterocyclic ring containing no unsaturated carbon atoms.
  • heterocycle and “heterocyclyl group” includes fused ring species including those having fused aromatic and non-aromatic groups.
  • the phrase also includes polycyclic ring systems containing a heteroatom and also includes heterocyclyl groups that have substituents, including but not limited to alkyl, halo, amino, hydroxy, cyano, carboxy, nitro, thio, or alkoxy groups, bonded to one of the ring members.
  • a heterocyclyl group as defined herein can be a heteroaryl group or a partially or completely saturated cyclic group including at least one ring heteroatom.
  • Heterocyclyl groups include, but are not limited to, pyrrolidinyl, furanyl,
  • benzothiophenyl benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, iso quinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.
  • heterocyclyl groups may be substituted.
  • Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, including but not limited to, rings containing at least one heteroatom which are mono, di, tri, tetra, penta, hexa, or higher-substituted with substituents such as those listed above, including but not limited to substituted alkyl where the substituent may be, for example, halo, amino, hydroxy, cyano, carboxy, azide, hydrazine, nitro, thio, or alkoxy; unsaturated alkyl having, for example, carbon- carbon double bonds and/or carbon-carbon triple bonds; and alkyl groups that are both unsaturated and substituted.
  • Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S.
  • a heteroaryl group designated as a C 2 -heteroaryl can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms and so forth.
  • a C 4 -heteroaryl can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, and so forth.
  • the number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms.
  • Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl,
  • benzimidazolyl azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, iso quinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, and quinazolinyl groups.
  • heteroaryl and “heteroaryl groups” include fused ring compounds such as wherein at least one ring, but not necessarily all rings, are aromatic, including tetrahydroquinolinyl, tetrahydroisoquinolinyl, indolyl and 2,3- dihydro indolyl.
  • the term also includes heteroaryl groups that have other groups bonded to one of the ring members, including but not limited to alkyl, halo, amino, hydroxy, cyano, carboxy, nitro, thio, or alkoxy groups.
  • Representative substituted heteroaryl groups can be substituted one or more times with substituents such as those listed herein.
  • the heterocycle group is a "nucleobase", where this term refers to a heterocyclic base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof.
  • a nucleobase can be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine,
  • the nucleobase is selected from adenine, guanine, uridine, and cytosine, and analogs of these nucleobases, such as those analogs disclosed herein.
  • Nucleobase residue includes nucleotides, nucleosides, fragments thereof, and related molecules having the property of binding to a complementary nucleotide. Deoxynucleotides and ribonucleotides, and their various analogs, are contemplated within the scope of this definition. Nucleobase residues may be members of oligomers and probes. "Nucleobase” and “nucleobase residue” may be used interchangeably herein and are generally synonymous unless context dictates otherwise.
  • the heterocycle may be denoted by the symbol "Bi_ 4 ", wherein the subscript indicates that the heterocycle may be any one of the four standard nucleobases, A, C, G, or T, or an analog thereof wherein one atom of a natural base is replaced with a different atom which typically allows for additional substitution on the nucleobase.
  • the heterocycle is a heterocyclic base.
  • Heterocyclic bases are well known in the art as being nitrogen containing ring structures bound though a glycosidic bond to a sugar moiety, such as a pentose (e.g., D-ribose and 2- deoxy-D-ribose), where the sugar moiety may be bonded to a phosphate, such as a monophosphate, a diphosphate and a triphosphate.
  • Exemplary heterocyclic bases are purines and pyrimidines. Exemplary purines ae adenine and guanine, while exemplary pyrimidines are cytosine, uracil and thymine.
  • the heterocyclic base includes substituted heterocyclic bases and analogs of a naturally occurring heterocyclic base wherein a native atom is replaced with a different atom (e.g., a nitrogen normally found in a heterocyclic base may be replaced with carbon, e.g., C-H or C-substituent).
  • a native atom is replaced with a different atom
  • a nitrogen normally found in a heterocyclic base may be replaced with carbon, e.g., C-H or C-substituent.
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom.
  • G 1 is a protecting group for a hydroxyl group that is bonded to a phosphorous atom, or in other words, G 1 is a protecting group that is bonded to an oxygen, where the oxygen is bonded to a phosphorous, so that the protecting group is protecting what would otherwise be a hydroxyl group bonded to the phosphorous atom.
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon.
  • G 3 is a protecting group that is bonded to an oxygen, where the oxygen is bonded to a carbon atoms, so that the protecting group is protecting what would otherwise be a hydroxyl group bonded to a carbon atom.
  • Protecting groups can render chemical functionality inert to specific reaction conditions and can be appended to and removed from such functionality in a molecule without substantially damaging the remainder of the molecule. Practitioners in the art would be familiar with suitable protecting groups for use in the synthetic methods of the invention. See, e.g. , Greene and Wuts, Protective Groups in Organic Synthesis, 2" ed., John Wiley k Sons, New York, 1991 and Peter G.M . Wuts, "Greene's Protective Groups in Organic Synthesis: Fifth Edition", Wiley, 2014.
  • a term refers to an integer selected from a range, then that term may be any integer within that range, including the ends of the range.
  • q is an integer selected from 2-10, then q can be any of 2, 3, 4, 5, 6, 7, 8, 9 and 10.
  • SS represents a solid support such as controlled pore glass (CPG).
  • CPG controlled pore glass
  • SS-L represents a solid support that is optionally bonded to a linking group, unless the presence of the linking groups is specifically excluded.
  • a linking group is optionally inserted between a solid support and the compound being synthesized by solid phase chemistry as disclosed herein.
  • the present disclosure provides a compound of the formula (1)
  • R 1 is selected from
  • R 2 is selected from hydrogen and C 1 -C 4 alkyl
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T);
  • R 13 is selected from a) an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and b) an alkyl group and an oxyalkyl group,
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 1 , R 2 , etc. may be used to further describe any of the compounds and synthetic methods disclosed herein which are described in terms of R 1 , R 2 , etc.
  • the R 1 group is a terminally-functionalized alkyl group, where the functional group is selected from, for example, carbon-carbon double bond, carbon-carbon triple bond, nucleophilic groups such as hydroxyl, thiol or amino, electrophilic groups such as halogen, or other reactive groups such as carboxyl, formyl (aldehyde) and hydroxyamino.
  • the alkyl group is a straight chain or a branched alkyl group having from 1 to about 20 carbon atoms (C C 20 alkyl or Ci_ 20 alkyl), or 1 to 12 carbons (Ci-Ci 2 alkyl or Ci_i 2 alkyl) or 1 to 8 carbon atoms (Ci-C 8 alkyl or Ci_ 8 alkyl) or 1 to 4 carbon atoms (Ci-C 4 alkyl or Ci_ 4 alkyl) or, in some embodiments, from 1 to 3 carbon atoms (Ci-C 3 alkyl or Ci_ 3 alkyl).
  • R 1 is a hydrocarbon group such as -(CH 2 ) q -C ⁇ CH and q is an integer selected from 2-10, e.g., R 1 may be 1-hexynyl of the formula
  • R 1 includes an electrophilic group as part of its structure, preferably the electrophilic group being at the terminus of the R 1 group.
  • R 1 includes a nucleophilic group as part of its structure, preferably the nucleophilic group being at the terminus of the R 1 group.
  • R 1 includes a carboxylic acid or an ester thereof as part of its structure, where the carboxylic acid or an ester thereof is preferably at the terminus of the R 1 group.
  • R 1 is an oxyalkyi group which is terminally functionalized, where an oxyalkyi group may also be called an oxyalkylene group, and refers to an alkyl group that incorporates one or more oxygen atoms in the form of ether groups.
  • the oxyalkyi group may be an oxyethyl (-0-CH 2 -CH 2 -) group or an oxypropyl (-O-CH2-CH2-CH2-) group, those being are exemplary oxyalkyi groups.
  • the oxyalkyi group of R 1 may be formed from one or a plurality of oxyalkyi units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 repeating units.
  • the functional group is selected from, for example, carbon-carbon double bond, carbon-carbon triple bond, nucleophilic groups such as hydroxyl, thiol or amino, electrophilic groups such as halogen, or other reactive groups such as carboxyl, formyl (aldehyde) and
  • the alkyl portion of the oxyalkyi is a straight chain or a branched alkyl group having from 1 to about 20 carbon atoms (C C 2 o alkyl or Ci-20 alkyl), or 1 to 12 carbons (C Ci 2 alkyl or Ci_i 2 alkyl) or 1 to 8 carbon atoms (C C 8 alkyl or Ci_ 8 alkyl) or 1 to 4 carbon atoms (C C 4 alkyl or Ci_ 4 alkyl) or, in some embodiments, has 1, 2 or 3 carbon atoms (C1-C3 alkyl or Ci_ 3 alkyl), or has 2 carbon atoms.
  • R 1 includes an electrophilic group as part of its structure, preferably the electrophilic group being at the terminus of the R 1 group. In one embodiment, R 1 includes a nucleophilic group as part of its structure, preferably the nucleophilic group being at the terminus of the R 1 group. In one embodiment, R 1 includes a carboxylic acid or an ester thereof as part of its structure, where the carboxylic acid or an ester thereof is preferably at the terminus of the R 1 group.
  • R 1 may be an alkyl group or an oxyalkyl group, either of which is terminally-functionalized, where the terminal functional group is selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl or ester thereof, formyl, hydroxylamino and halogen.
  • the R 2 group is selected from hydrogen and Ci-C 4 alkyl. In one
  • R 2 is hydrogen in each of the embodiments and embodiment combinations as disclosed herein. In another embodiment, R 2 is Ci, i.e., methyl.
  • R 5 is selected from H and G 1 ; and G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom.
  • R 3 represents R 5
  • R 5 is H.
  • R 3 represents R 5
  • R 5 is a protecting group G 1 .
  • R 3 is -[Pn-0] m -R 5 .
  • the present disclosure provides each of the following exemplary structures (1A)-(1K):
  • the compounds of the present disclosure may have, for example, multiple phosphate groups or phosphate ester groups.
  • each of the phosphorous atoms is in the +5 valence state except for the terminal phosphorous atom which is in the +3 valence state, and R 5 may be hydrogen or G 1 , independently selected at each occurrence.
  • R 5 may be hydrogen or G 1 , independently selected at each occurrence.
  • R is a cyclic group of the formula R R .
  • the R group is an acyclic groups of the In
  • R 6 is a heterocycle, where in optional embodiments R D is a nucleobase or a heterocyclic base that may be substituted with R 13 as defined herein.
  • Exemplary R 6 nucleobases are the Bi_ 4 nucleobases, where this term refers to a nucleobase selected from an adenosine analog, a guanosine analog, a uridine analog and a cytidine analog.
  • Bi_ 4 may refer to an adenosine
  • R is selected from an alkyl group and an oxyalkyl group, either of which is terminally functionalized.
  • the term "either of which” as used herein refers to the alkyl group and the oxyalkyl group.
  • Exemplary functional groups are selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen.
  • the terminal functional group of R 13 and the terminal functional group of R 1 are the same functional group, for example, the terminal functional group of both R 1 and R 13 is a carbon-carbon triple bond such as shown in compound (11) disclosed elsewhere herein.
  • the R 13 group may be a terminally-functionalized alkyl group, where the functional group may be selected from carbon-carbon double bond, carbon- carbon triple bond, nucleophilic groups such as hydroxyl, thiol or amino, electrophilic groups such as halogen, or other reactive groups such as carboxyl, formyl (aldehyde) and hydroxyamino.
  • the alkyl group is a straight chain or a branched alkyl group having from 1 to about 20 carbon atoms (C C 2 o alkyl or Ci_ 2 o alkyl), or 1 to 12 carbons (C Ci 2 alkyl or Ci_ i2 alkyl) or 1 to 8 carbon atoms (Ci-C 8 alkyl or Ci-8 alkyl) or 1 to 4 carbon atoms (Ci-C 4 alkyl or Ci_ 4 alkyl) or, in some embodiments, from 1 to 3 carbon atoms (C C 3 alkyl or Ci_ 3 alkyl).
  • R 1 is a hydrocarbon alkyl group that is or includes the moiety -(CH 2 ) r -C ⁇ CH and r is an integer selected from 2-10, e.g., R 13 is or comprises 1-hexynyl of the formula - CH2CH2CH2CH 2 C ⁇ CH.
  • R 13 includes an electrophilic group as part of its structure, preferably the electrophilic group being at the terminus of the R 13 group.
  • R 13 includes a nucleophilic group as part of its structure, preferably the nucleophilic group being at the terminus of the R 13 group.
  • R 13 includes a carboxylic acid or an ester thereof as part of its structure, where the carboxylic acid or an ester thereof is preferably at the terminus of the R 13 group.
  • the terminal reactive group of R 1 is identical to the terminal reactive group of R 13 , e.g., both R 1 and R 13 terminate in a carbon-carbon triple bond, e.g., each may terminate in a -CH2-CH2-CH2- CH 2 -C ⁇ CH group.
  • R 13 is or comprises an omega-functionalized C 6 - Ci 6 hydrocarbon or an omega-functionalized C 6 -Ci 6 alkyl.
  • the R 13 group may be a terminally- functionalized oxyalkyi group, where the functional group may be selected from carbon-carbon double bond, carbon-carbon triple bond, nucleophilic groups such as hydroxyl, thiol or amino, electrophilic groups such as halogen, or other reactive groups such as carboxyl, formyl (aldehyde) and hydroxyamino.
  • the oxyalkyi group incorporates a straight chain or a branched alkyl group having from 1 to about 20 carbon atoms (C C 2 o alkyl or Ci_ 20 alkyl), or 1 to 12 carbons (C Ci 2 alkyl or Ci_ 12 alkyl) or 1 to 8 carbon atoms (Ci-C 8 alkyl or Ci_ 8 alkyl) or 1 to 4 carbon atoms (C C 4 alkyl or Ci_ 4 alkyl) or, in some embodiments, from 1 to 3 carbon atoms (C1-C3 alkyl or Ci_3 alkyl), while in one embodiment the alkyl group of each oxyalkyi unit has 2 carbons, and in another embodiment the alkyl group of each oxyalkyi unit has 3 carbons.
  • R 13 includes an electrophilic group as part of its structure, preferably the electrophilic group being at the terminus of the R group.
  • R 13 includes a nucleophilic group as part of its structure, preferably the nucleophilic group being at the terminus of the R 13 group.
  • R 13 includes a carboxylic acid or an ester thereof as part of its structure, where the carboxylic acid or an ester thereof is preferably at the terminus of the R 13 group.
  • the terminal reactive group of R 1 is identical to the terminal reactive group of R 13 , e.g., both R 1 and R 13 terminate in a carbon-carbon triple bond.
  • R 4 is a heterocycle or nucleobase which includes R 13 as part of its structure, where R 13 is selected from omega-functionalized C 6 -Ci 6 hydrocarbons or omega-functionalized C 6 -Ci 6 alkyls.
  • R 13 is selected from omega-functionalized C 6 -Ci 6 hydrocarbons or omega-functionalized C 6 -Ci 6 alkyls.
  • An exemplary R 13 group is -C ⁇ C- (CH 2 ) 4 -C ⁇ CH in which case the omega functional group is a carbon-carbon triple bond.
  • R 4 is a heterocycle or nucleobase which includes R 13 as a substituent, where R 13 is an alkyl group having a terminal carbon- carbon triple bond, and R 1 is an alkyl group having a terminal carbon-carbon triple bond, so that the compound of form ula (1) may be a bis-alkyne deoxynucleoside polyphosphoramidate, e.g., a bis-alkyne deoxynucleoside triphosphoramidate.
  • Such a bis-alkyne structure is a particularly useful compound to react with a tether precursor having terminal azide groups, i.e., N 3 -tether-N 3 , where the product of such a reaction comprises triazole groups that link the two ends of the tether (via LG1 and LG2, each of LG1 and LG2 being a triazole group) to a deoxynucleoside polyphosphoramidate such as a deoxynucleoside triphosphoramidate.
  • a tether precursor having terminal azide groups i.e., N 3 -tether-N 3
  • the product of such a reaction comprises triazole groups that link the two ends of the tether (via LG1 and LG2, each of LG1 and LG2 being a triazole group) to a deoxynucleoside polyphosphoramidate such as a deoxynucleoside triphosphoramidate.
  • R 13 is selected from omega-functionalized C 6 -Ci 6 hydrocarbons or omega-functionalized C 6 -Ci 6 alkyls.
  • An exemplary R 13 group is -C ⁇ C- (CH 2 ) 4 -C ⁇ CH in which case the omega functional group is a carbon-carbon triple bond.
  • the R 7 group is selected from hydrogen, -CH 2 -halogen, C C 4 alkyl, hydroxyl and -CH 2 -OR 10 .
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl and -CH 2 -OR 10 .
  • the R group is -OR or -O-SSL.
  • SSL and SS-L designate a solid support (SS) that is optionally bound to a linking (also referred to as a linker) group (L), where the linker group joins the solid support through an oxygen atom as shown, to the remainder of the molecule.
  • R 9 group is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 - R 12 where R 10 and R 12 form a direct bond. I n one embodiment, R 9 is hydrogen. In one embodiment, R 7 is -CH 2 -OR 10 and R 9 is -CH 2 -R 12 where R 10 and R 12 form a direct bond.
  • the R 11 group is H or a protecting group G 3 ; where G 3 is a protecting
  • R is hydrogen.
  • R is a protecting group, G
  • the G group is selected from oxygen, sulfur and CH 2 .
  • G 2 is oxygen. I n one embodiment, G 2 is sulfur. In one embodiment, G 2 is
  • the present disclosure provides a compound of the formula
  • R 1 , R 2 , R 3 and R 4 are defined herein, and embodiments for R 1 , R 2 , R 3 and R 4 are provided, including embodiments for R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and R 13 , G 1 , G 2 , G 3 , Pn, m, q and r.
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and R 13 , G 1 , G 2 , G 3 , Pn, m, q and r In describing compound of formula (I), any two, or any three, or any four, or any five, or more than five of these various embodiments may be combined.
  • the present disclosure provides a compound of the formula 2a)
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and
  • R 12 form a direct bond
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 2 is hydrogen; and independently G 2 is oxygen.
  • the present disclosure provides a com ound of formula (2b)
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and
  • R form a direct bond
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 9 is hydrogen.
  • G 2 is oxygen and/or R 2 is hydrogen.
  • the present disclosure provides a compound of formula (2c)
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbo carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the present disclosure also provides compounds corresponding to formulae (2a), (2b) and (2c) however the pentavalent phosphorous atom is trivalent. I n other words, the present disclosure provides compounds of formula (2d), (2e) and 2f) where groups R 1 , etc. are as defined above.
  • R 1 is a terminally-functionalized alkyl group, where the functional group is carbon-carbon triple bond, e.g., R 1 is -(CH 2 ) q -C ⁇ C and q is an integer selected from 2-10; and/or R 5 in at least one occurrence is hydrogen, and/or R 5 in at least one occurrence is G 1 ; and/or R 6 is a nucleobase or R 6 is a heterocyclic base; and/or R 7 is hydrogen; and/or R 8 is OH or R 8 comprises a solid support.
  • the present disclosure provides a compound of the formula (3a
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom; G is selected from oxygen, sulfur and CH 2 ; and
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 2 is hydrogen; and independently G 2 is oxygen.
  • the present disclosure provides compound of formula (3b)
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 9 is hydrogen.
  • G 2 is oxygen and/or R 2 is hydrogen.
  • the present disclosure provides a com ound of formula (3c)
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, hydroxyl and Ci-C 4 alkyl;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the present disclosure also provides compounds corresponding to formulae (3a), (3b) and (3c) however some of the pentavalent phosphorous atoms are trivalent.
  • the present disclosure provides compounds of formula (3d), (3e) and (3f) where groups R 1 , etc. are as defined above.
  • R 1 is a terminally-functionalized alkyl group, where the functional group is carbon-carbon triple bond, e.g., R 1 is -(CH 2 ) q -C ⁇ C and q is an integer selected from 2-10; and/or G 2 is oxygen; and/or R 5 in at least one occurrence is hydrogen, and/or R 5 in at least one occurrence is G 1 ; and/or R 6 is a nucleobase or R 6 is a heterocyclic base; and/or R 7 is hydrogen; and/or R 8 is OH or R 8 comprises a solid support.
  • R 1 is a terminally-functionalized alkyl group, where the functional group is carbon-carbon triple bond, e.g., R 1 is -(CH 2 ) q -C ⁇ C and q is an integer selected from 2-10; and/or G 2 is oxygen; and/or R 5 in at least one occurrence is hydrogen, and/or R 5 in at least one occurrence is G 1 ; and/or R 6 is a
  • the present disclosure provides a compound of the formula (4a) wherein: G 1 is H or a protecting group; R 6 is a heterocycle; R 8 is selected from OR 11 and O-SS; R 11 is selected from H and G 3 ; G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom; and SS represents a solid support optionally bound to the 0 of O-SS via a linking group (L).
  • R 8 is hydroxyl.
  • R 6 is a nucleobase.
  • R 6 is a heterocyclic base.
  • the protecting group G 1 may be a
  • R 6 is a heterocycle
  • R 8 is - OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker
  • R 11 is selected from H and G 3
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom
  • SS represents a solid support optionally bound to the 0 of O-SS via a linking group (L).
  • R 8 is hydroxyl.
  • R 8 is protected hydroxyl.
  • R 8 includes a solid support.
  • R 6 is a nucleobase.
  • R 6 is a uridine analog.
  • An exemplary uridine analog is shown in the compound of the formula
  • G 1 is shown as cyanoethyl however other protecting groups may be substituted for cyanoethyl
  • R is shown as -C ⁇ C-(CH 2 ) 4 -C ⁇ CH however other omega-functionalized C 6 -Ci 6 hydrocarbon groups may be substituted for -C ⁇ C-(CH 2 ) 4 -C ⁇ CH
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker; R 11 is selected from H and G 3 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom; and
  • SS represents a solid support optionally bound to the 0 of O-SS via a linking group.
  • R 8 is hydroxyl. In one embodiment, R 8 is protected hydroxyl. In yet another embodiment, R 8 includes a solid support. As another example, in one embodiment R 6 is a cytidine analog. An exemplary cytidine analog is shown in the compound of the formula (4d),
  • G 1 is shown as cyanoethyl however other protecting groups may be substituted for cyanoethyl;
  • R is shown as -C ⁇ C-(CH 2 ) 4 -C ⁇ CH however other omega-functionalized C 6 -Ci 6 hydrocarbon groups may be substituted for -C ⁇ C-(CH 2 ) 4 -C ⁇ CH;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker; R 11 is selected from H and G 3 ; and G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 8 is hydroxyl.
  • R 8 is protected hydroxyl.
  • R 8 includes a solid support.
  • R 6 is an adenosine analog. An exemplary adenosine analog is shown in the compound of the formula (4e),
  • G 1 is shown as cyanoethyl however other protecting groups may be substituted for cyanoethyl;
  • R is shown as -C ⁇ C-(CH 2 ) 4 -C ⁇ CH however other omega-functionalized C 6 -Ci 6 hydrocarbon groups may be substituted for -C ⁇ C-(CH 2 ) 4 -C ⁇ CH;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker; R 11 is selected from H and G 3 ; and G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 8 is hydroxyl.
  • R 8 is protected hydroxyl.
  • R 8 includes a solid support.
  • R 6 is a guanosine analog. An exemplary guanosine analog is shown in the compound of the formula (4f),
  • G 1 is shown as cyanoethyl however other protecting groups may be substituted for cyanoethyl
  • R 13 is shown as -C ⁇ C-(CH 2 ) 4 -C ⁇ CH however other omega-functionalized C 6 -Ci 6 hydrocarbon groups may be substituted for -C ⁇ C-(CH 2 ) 4 -C ⁇ CH
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker; R 11 is selected from H and G 3 ; and G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 8 is hydroxyl.
  • I n one embodiment, R 8 is protected hydroxyl.
  • R 8 includes a solid support.
  • the present disclosure also provides a cyclic phosphite of the formula (5) and salts thereof,
  • R 1 is an alkyl group or an oxyalkyl group, either of which is terminally- functionalized, where the terminal functional group is selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl or ester thereof, formyl, hydroxylamino and halogen.
  • the terminal functional group of R 1 may be carbon-carbon double bond; and/or it may be carbon-carbon triple bond; and/or it may be hydroxyl; and/or it may be amine; and/or it may be thiol; and/or it may be carboxyl or ester thereof; and/or it may be formyl; and/or it may be hydroxylamino; and/or it may be halogen.
  • R 1 when R 1 is an alkyl group and the functional group is a carbon- carbon triple bond, R 1 may be -(CH 2 ) q -C ⁇ CH where -(CH 2 ) q is the alkyl group, which might also be referred to as an alkylene group, and q is an integer selected from 2-10, e.g., R 1 is 1-hexynyl of the formula -CH 2 CH 2 CH 2 CH 2 C ⁇ CH.
  • R 1 includes an electrophilic group.
  • R 1 includes a nucleophilic group.
  • R 1 includes a carboxylic acid or an ester thereof.
  • R 1 is an alkyl group which is terminally-functionalized.
  • R 1 is an oxyalkyl group which is terminally functionalized, where an oxyalkyl group may also be called an oxyalkylene group, and refers to an alkyl group that incorporates one or more oxygen atoms in the form of ether groups.
  • Oxyethyl (- 0-CH 2 -CH 2 -) groups and oxypropyl (-0-CH 2 -CH 2 -CH 2 -) groups are exemplary oxyalkyl groups.
  • the oxyalkyl group of R 1 may be formed from one or a plurality of oxyalkyl units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 repeating units.
  • the present invention provides a process for forming an N-phosphoroamidate diester (110) as illustrated in Scheme 1.
  • Scheme 1 N-phosphoroamidate diester (110)
  • the present disclosure provides a process of forming a phosphoromonoamidate diester 110 from a phosphite triester compound (100) and an azide compound (105),
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and C 1 -C 4 alkyl
  • R 4 is selected from and
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and
  • R 12 form a direct bond
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the protected phosphite (100) may be synthesized from the corresponding N,N-diisopropylphosphoramidite (90), where (90) may be obtained from commercial sources (for example, from Chemgenes of
  • Activators sometimes referred to as coupling activators, are known in the art of phosphoramidite chemistry and oligonucleotide synthesis, where other suitable activators include 5-ethylthio-lH- tetrazole, 5-benzylthio-lH-tetrazole and 4,5-dicyanoimidazole, each available from, e.g., Glen Research (Sterling, VA). See also, for example, Dahl, B.H., et al. Nucleic Acids Res (1987) 15:1729-43; Vargeese, C. et al., Nucl. Acids Res. (1998) 26 (4):1046-1050; and Berner, S., Nucleic Acids Res.
  • Benzimidazolium triflate may also be used as an activator, see, e.g., Hayakawa Y., et al., J. Org. Chem. (1996) 61:7996- 7997.
  • the present disclosure provides a process wherein compound 100 is prepared from reaction of compound 90 and an alcohol of formula HO-alkyl where the reaction is conducted in the presence of an activator.
  • the azide (105) from Scheme 1 may be prepared from the corresponding iodo compound (85), which in turn may be prepared from the corresponding protected hydroxyl compound (80) as illustrated in Scheme 3.
  • the protected hydroxyl compound (80) may be converted to the corresponding iodo compound (85) by a two-step reaction.
  • the protecting group G 3 is removed under conditions that are appropriate for that particular protecting group.
  • the protecting group G 3 is dimethoxytrityl ether (DMTr) then G 3 can be removed by treatment with 3% trichloroacetic acid (TCA) in a suitable solvent such as methylene chloride to provide the free hydroxyl compound, i.e., G 3 is hydrogen.
  • DMTr dimethoxytrityl ether
  • TCA 3% trichloroacetic acid
  • the free hydroxyl compound is reacted with methyltriphenoxyphosphonium iodide in a suitable solvent such as dimethylformamide to provide the corresponding iodo compound (85).
  • a suitable solvent such as dimethylformamide
  • the iodo compound (85) may be readily converted to the corresponding azide (105) by treatment with sodium azide in a suitable solvent, such as dimethylformamide.
  • the present disclosure provides a process wherein compound 80 is converted to compound 85 and compound 85 is converted to compound 105
  • G 3 is removed under conditions that are appropriate for that particular protecting group to provide the corresponding free hydroxyl compound, i.e., G 3 is hydrogen; and the free hydroxyl compound is reacted with methyltriphenoxyphosphonium iodide in a suitable solvent to provide the
  • the present invention provides a process for forming a phosphate protected N-phosphoroamidate-monoester diphosphate (120) as illustrated in Scheme 4.
  • a suitably protected N-phosphoroamidate diester (110) is reacted with a base such as l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in the presence of a silylating agent such as ⁇ , ⁇ -bis-trimethylsilylacetamide (BSA) to form a first intermediate (not shown in Scheme 4) which is subsequently reacted, optionally and preferably without isolation, with a phosphorylating agent such as the phosphorylating phosphoramidite (115, available commercially from, e.g., Chemgenes, Wilmington, MA, USA) as shown in Scheme 4, in the presence of an activator such as 5-(ethylthio)- lH-tetrazole (ETT) to form a second intermediate (not shown in Scheme 4) which is subsequently reacted, optionally and preferably without isolation, with an oxidizing agent such as an organic peroxide such as t-butylhydroper
  • the present disclosure provides a process of forming a phosphate protected N-phosphoroamidate-monoester disphosphate (120) from a phosphoroamidate diester compound (110) and a phosphorylating
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 4 is selected from
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the present invention provides a process for forming a phosphate protected N-phosphoroamidate-monoester triphosphate (125) as illustrated in Scheme 5.
  • a suitably protected phosphate protected N- phosphoroamidate-monoester diphosphate (120) is reacted with a base such as DBU and a silylating agent such as BSA to form a first intermediate (not shown in Scheme 5) which is subsequently reacted, optionally and preferably without isolation, with a phosphorylating agent such as the phosphorylating phosphoramidite (115) as shown in Scheme 5, in the presence of an activator such as ETT to form a second intermediate (not shown in Scheme 5) which is subsequently reacted, optionally and preferably without isolation, with an organic peroxide such as t-butylhydroperoxide in a suitable solvent such as methylene chloride to form the phosphate protected N- phosphoroamidate-monoester triphosphate (125).
  • a base such as DBU and a silylating agent such as BSA
  • a phosphorylating agent such as the phosphorylating phosphoramidite (115) as shown in
  • the present disclosure provides a process of forming a phosphate protected N-phosphoroamidate-monoester triphosphate (125) from a phosphate protected N-phosphoroamidate-monoester diphosphate compound (120) and a phosphorylating phosphoramidite compound (115),
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the phosphate protected N-phosphoroamidate-monoester triphosphate (125), which may be prepared as shown in Scheme 5, comprises protecting groups G 1 , and may include a solid support SS through R 8 of R 4 .
  • phosphate protected N-phosphoroamidate-monoester triphosphate (125) is exposed to conditions suitable for removing the protecting groups G 1 and cleaving the linker of the solid support if present. The choice of suitable conditions will depend on the identity of the protecting groups and the linking group that have been employed to make (125).
  • the protecting groups are base labile, e.g., trimethylsilyl groups
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR ;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • the present invention provides a process for forming a compound (130) by deprotecting a compound (125), where compound (125) may be synthesized as shown in Scheme 5.
  • the present disclosure provides an alternative process for forming a compound (130) which is illustrated in Schemes 6-8.
  • a cyclic phosphite (145) is prepared from an alcohol R 1 -OH (compound 140), e.g., 5-hexyn-l-ol, and commercially available 2-chloro-4H-l,3,2- benzodioxaposphorin-4-one (compound 135) by combining these reactants in a suitable solvent such a dimethylformamide and in the presence of a suitable base such as tributylamine, at a suitable reaction temperature such as about room temperature, for a suitable period of time such as for about 5-60 minutes, to prepare compound 145, e.g., salicyl-(5-hexyn-l-yl) phosphite.
  • suitable reaction conditions are disclosed in, e.g., Ludwig and Eckstein, J. Org. Chem. 56:1777-1783 (1991).
  • the product (145) from Scheme 6 may be added to commercially available 0.5M bis-tributylammonium pyrophosphate (150) in a suitable solvent, such as dimethylformamide, at a suitable temperature such as about room temperature, and for a suitable period of time, such as for 5-60 minutes, to provide the salt of the cyclotriphosphite compounds (155), as illustrated in Scheme 7.
  • a suitable solvent such as dimethylformamide
  • the cyclotriphosphite (155) may be reacted with previously described azide (105) under suitable reaction conditions such as in a suitable solvent such as dimethylsulfoxide, at a suitable reaction temperature such as about 55°C, and for a suitable time period such as about 24-36 hours, as shown in Scheme 8,
  • R 1 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR ;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R is hydrogen
  • the present disclosure provides a process for forming a N-phosphoroamidate-monoester triphosphate (160) from a
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 6 is a heterocycle, optionally substituted with R 13 , where R 13 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • the R 8 group may be or includes a solid support, so that, for example, one or more of, and preferably all of, the conversion of compound (80) to compound (85), the conversion of compound (85) to (105), the conversion of compound (105) to compound (110), and the conversion of compound (155) to compound (160) is performed using solid phase synthesis techniques.
  • SBX SBX Expansion
  • Xpandomers are generated from polymerization of non-natural nucleotide analogs, termed XNTPs, which are expandable, 5' triphosphate modified nucleotide substrates compatible with template dependent enzymatic polymerization.
  • An XNTP has two distinct functional regions; namely, a nucleoside triphosphoramidate and a tether that is attached within each nucleoside triphosphoramidate at positions that allow for controlled expansion by intra-nucleotide cleavage of the
  • the XNTP 100 is comprised of nucleobase triphosphoramidate 110 with linker arm moieties 120A (which is shown as a C 4 hydrocarbon chain that is a part of R 1 as disclosed herein) and 120B (which is shown as a C 6 hydrocarbon chain that is part of R 13 as disclosed herein) separated by selectively cleavable phosphoramidate bond 130.
  • linker 120A and 120B attaches to one end of tether 140 via a linking group (LG), as disclosed in U.S. Patent No. 8,324,360 to Kokoris et al., which is herein incorporated by reference in its entirety.
  • Tethers are polymers or molecular constructs having a generally linear dimension and with an end moiety at each of two opposing ends which are attached to the nucleobase
  • XNTPs have a "constrained configuration" and an "expanded configuration".
  • the constrained configuration is found in XNTPs and in the daughter strand.
  • the constrained configuration of the XNTP is the precursor to the expanded configuration, as found in Xpandomer products. The transition from the constrained configuration to the expanded configuration occurs upon scission of the P- N bond 130 of the phosphoramidate within the primary backbone of the daughter strand.
  • Tethers are joined to the nucleoside triphosphoramidate at linking group 150A and 150B, wherein a first tether end is joined to the heterocycle (represented in the Figure by the symbol "Bi_ 4 ", wherein the subscript indicates that the heterocycle may be any one of the four standard nucleobases, A, C, G, or T) and the second tether end is joined to the alpha phosphate of the nucleobase backbone.
  • the amino linker on 7-(octa-l,7-dinyl)-7-deaza- 2'-dATP can be used as a first tether attachment point
  • a mixed backbone linker such as the non-bridging modification (N-l-aminoalkyl) phosphoramidate
  • N-l-aminoalkyl phosphoramidate can be used as a second tether attachment point.
  • suitable coupling chemistries known in the art may be used to form the final XNTP substrate product, for example, tether conjugation may be accomplished through a triazole linkage.
  • the present disclosure provides a process in which an N- phosphoroamidate-monoester triphosphate (160) as described previously is reacted with a tether precursor of the formula X-T-X where X represents a reactive functional group that is reactive with the terminating functional groups of R 1 and R 13 , so as to form linker groups LGl and LG2.
  • X-T-X may be a bis-azide compound of the formula N 3 -T-N 3 , and the terminating functional groups of R 1 and R 13 are alkyne groups, so as to form triazole groups LGl and LG2.
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LGl), the LGl bonded to a tether (T);
  • R 2 is selected from hydrogen and Ci-C 4 alkyl;
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, the heterocycle comprising a substituent R 13 , where R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T);
  • R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T);
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 1 is an alkyl group which terminates in a linker group (LG1), the LG1 bonded to a tether (T);
  • R 1 is an oxyalkyi group which terminates in a linker group (LG1), the LG1 bonded to a tether (T);
  • R 2 is hydrogen
  • R 3 is R 5 ;
  • R 6 is a heterocycle, the heterocycle comprising a substituent R 13 , where
  • R 13 is an alkyl group which terminates in a linker group (LG2), the LG2 bonded to the tether (T);
  • R 6 is a heterocycle, the heterocycle comprising a substituent R 13 , where R 13 is an oxyalkyi group which terminates in a linker group (LG2), the LG2 bonded to the tether (T)
  • R 7 is hydrogen
  • R 7 is -CH 2 -halogen
  • R 7 is Ci-C 4 alkyl
  • R 7 is hydroxyl
  • R 7 is -CH 2 -OR 10 ;
  • R 8 is -O-L-SS where L-SS represents a solid support optionally bound to
  • R 9 is -CH 2 -R 12 where R 10 and R 12 form a direct bond
  • (x) G 1 is a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • LG1 and LG2 are each triazole.
  • the monomeric XNTP substrate construct is polymerized on the extendable terminus of the nascent daughter strand by a process of template- directed polymerization using a single-stranded template as a guide. Generally, this process is initiated from a primer and proceeds in the 5' to 3' direction. Generally, a DNA polymerase or other polymerase is used to form the daughter strand, and conditions are selected so that a complementary copy of the template strand is obtained.
  • tether or "tether member” refers to a polymer or molecular construct having a generally linear dimension and with an end moiety at each of two opposing ends.
  • a tether is attached to a substrate with a linkage in at least one end moiety to form a substrate construct.
  • the end moieties of the tether may be connected to cleavable linkages to the substrate or cleavable intra-tether linkages that serve to constrain the tether in a "constrained configuration".
  • each end moiety has an end linkage that couples directly or indirectly to other tethers.
  • the coupled tethers comprise the constrained Xpandomer that further comprises the daughter strand.
  • Tethers have a "constrained configuration" and an "expanded configuration".
  • the constrained configuration is found in substrate constructs and in the daughter strand.
  • the constrained configuration of the tether is the precursor to the expanded configuration, as found in Xpandomer products.
  • the transition from the constrained configuration to the expanded configuration results cleaving of selectively cleavable bonds that may be within the primary backbone of the daughter strand or intra-tether linkages.
  • Tether in a constrained configuration is also used where a tether is added to form the daughter strand after assembly of the "primary backbone".
  • Tethers can optionally comprise one or more reporters or reporter constructs along its length that can encode sequence information of substrates.
  • the tether provides a means to expand the length of the Xpandomer and thereby lower the sequence information linear density
  • Tether constructs are tethers or tether precursors composed of one or more tether segments or other architectural components for assembling tethers such as reporter constructs, or reporter precursors, including polymers, graft copolymers, block copolymers, affinity ligands, oligomers, haptens, aptamers, dendrimers, linkage groups or affinity binding group (e.g., biotin).
  • Tether element or "tether segment” (T) is a polymer having a generally linear dimension with two terminal ends, where the ends form end-linkages (LG1 and LG2) for concatenating the tether elements.
  • a precursor to such a tether element may have the formula X-T-X wherein T represents the tether element and X is a reactive functional group that will react so as to form end-linkages LG1 and LG2, where LG1 and LG2 are also joined to a nucleobase triphosphoramidate, and are shown in the FIGURE as 150B and 150A, respectively.
  • Tether elements may be segments of tether constructs.
  • Such polymers can include, but are not limited to: polyethylene glycols, polyglycols, polypyridines, polyisocyanides, polyisocyanates,
  • poly(triarylmethyl)methacrylates polyaldehydes, polypyrrolinones, polyureas, polyglycol phosphodiesters, polyacrylates, polymethacrylates, polyacrylamides, polyvinyl esters, polystyrenes, polyamides, polyurethanes, polycarbonates, polybutyrates, polybutadienes, polybutyrolactones, polypyrrolidinones,
  • polyvinylphosphonates polyacetamides, polysaccharides, polyhyaluranates, polyamides, polyimides, polyesters, polyethylenes, polypropylenes, polystyrenes, polycarbonates, polyterephthalates, polysilanes, polyurethanes, polyethers, polyamino acids, polyglycines, polyprolines, N-substituted polylysine, polypeptides, side-chain N-substituted peptides, poly-N-substituted glycine, peptoids, side-chain carboxyl-substituted peptides, homopeptides, oligonucleotides, ribonucleic acid oligonucleotides, deoxynucleic acid oligonucleotides, oligonucleotides modified to prevent Watson-Crick base pairing, oligonucleotide analogs, polycytidylic acid, polyadeny
  • Reporter element is a signaling element, molecular complex, compound, molecule or atom that is also comprised of an associated "reporter detection characteristic".
  • Other reporter elements include, but are not limited to, FRET resonant donor or acceptor, dye, quantum dot, bead, dendrimer, upconverting fluorophore, magnet particle, electron scatterer (e.g., boron), mass, gold bead, magnetic resonance, ionizable group, polar group, hydrophobic group.
  • fluorescent labels such as but not limited to, ethidium bromide, SYBR Green, Texas Red, acridine orange, pyrene, 4-nitro-l,8-naphthalimide, TOTO-1, YOYO-1, cyanine 3 (Cy3), cyanine 5 (Cy5), phycoerythrin, phycocyanin, allophycocyanin, FITC, rhodamine, 5(6)- carboxyfluorescein, fluorescent proteins, DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6- tetramethylpiperidine), dinitrophenyl, acridines, coumarins, Cy3 and Cy5 (Biological Detection Systems, Inc.), erytrosine, coumaric acid, umbelliferone, texas red r
  • mass tags such as, for example, pyrimidines modified at the C5 position or purines modified at the N7 position, wherein mass modifying groups can be, for examples, halogen, ether or polyether, alkyl, ester or polyester, or of the general type XR, wherein X is a linking group and R is a mass-modifying group, chemiluminescent labels, spin labels, enzymes (such as peroxidases, alkaline phosphatases, beta-galactosidases, and oxidases), antibody fragments, and affinity ligands (such as an oligomer, hapten, and aptamer).
  • mass modifying groups can be, for examples, halogen, ether or polyether, alkyl, ester or polyester, or of the general type XR, wherein X is a linking group and R is a mass-modifying group, chemiluminescent labels, spin labels, enzymes (such as peroxidases, alkaline phosphatases, beta-gal
  • Association of the reporter element with the tether can be covalent or non-covalent, and direct or indirect.
  • Representative covalent associations include linker and zero- linker bonds. I ncluded are bonds to the tether backbone or to a tether-bonded element such as a dendrimer or sidechain.
  • Representative non-covalent bonds include hydrogen bonds, hydrophobic bonds, ionic bonds, pi-bond ring stacking, Van der Waals interactions, and the like.
  • Ligands for example, are associated by specific affinity binding with binding sites on the reporter element. Direct association ca n take place at the time of tether synthesis, after tether synthesis, and before or after Xpandomer synthesis.
  • a "reporter” is composed of one or more reporter elements. Reporters include what are known as “tags” and “labels.” The probe or nucleobase residue of the Xpandomer can be considered a reporter. Reporters serve to parse the genetic information of the target nucleic acid.
  • Reporter construct comprises one or more reporters that can produce a detectable signal(s), wherein the detectable signal(s) generally contain sequence information. This signal information is termed the "reporter code” and is
  • a reporter construct may also comprise tether segments or other a rchitectural components including polymers, graft copolymers, block copolymers, affinity ligands, oligomers, haptens, aptamers, dendrimers, linkage groups or affinity binding group (e.g., biotin).
  • tether segments or other a rchitectural components including polymers, graft copolymers, block copolymers, affinity ligands, oligomers, haptens, aptamers, dendrimers, linkage groups or affinity binding group (e.g., biotin).
  • enantiomers/diastereomers may be obtained by methods known to those skilled in the art.
  • the starting materials and various reactants utilized or referenced in the examples may be obtained from commercial sources, or are readily prepared from commercially available organic compounds, using methods well-known to one skilled in the art.
  • HBTU (l/-/-benzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (EMD Millipore, Billerica, MA). Methyltriphenoxyphosphonium iodide (Toronto Research Chemicals, Toronto, ON CANADA). 5'-0-Dimethoxytrityl-5-(octa-l,7-diynyl)-2'-deoxyuridine (3) (ChemBiotech, M unster, Germany). 0.5M Bis-tributylammonium pyrophosphate in DM F (15, GL Synthesis Inc., Worcester, MA).
  • LiCI lithium chloride
  • DI EA diisopropylethylamine
  • DBU l,8-diazabicyclo[5.4.0]undec-7-ene
  • TBHP t-butylhydroperoxide
  • DCM diichloromethane
  • ACN acetonitrile
  • DMF dimethylformamide
  • 2-chloro-4H- l,3,2-benzodioxaphosphorin-4-one salicyl chlorophosphite
  • 5-hexyn-l-ol (13) may also be obtained from Sigma, St. Louis, MO.
  • Solvents are anhydrous and packaged in SureSealTM containers or equivalent. 2M triethylammonium acetate,
  • Pac 2 0 Cap A (5% (w/v) phenoxyacetic anhydride: 10% pyridine in THF
  • Pac 2 0 Cap B (16% 1-methylimidazole in THF may each be obtained from Glen Research, Sterling, VA.
  • High performance liquid chromatography is performed on a ProStar HelixTM HPLC system from Agilent Technologies, Inc. (Santa Clara, CA) consisting of two pumps (ProStar 210 Solvent Delivery Modules) with 10 ml titanium pump heads, a column oven (ProStar 510 Air Oven), a UV detector (ProStar 320 UV/Vis Detector) set at 292 nm. The system is controlled by Star Chromatography Workstation Software (Version 6.41).
  • the column used is a Cadenza CD-C18, 3 ⁇ (4.6mm x 150mm) equipped with an in-line Cadenza Guard Column System for CD-C18 (2.0mm X 5mm) both from I mtakt USA (Portland, OR).
  • the buffers used are: Buffer A (100 mM triethylammonium acetate, pH 7.0) and Buffer B (100 mM triethylammonium acetate, pH 7.0 with 95% by volume acetonitrile).
  • ESI Mass spectrometry was done by Numega Resonance Lab (San Diego, CA). Mass specs on CPG-bound intermediates were performed on the products recovered after deprotection and cleavage off of the solid support. All ESI MS (positive mode) were consistent with the fully deprotected structures.
  • Synthetic Scheme A provides an outline of a methodology according to the present disclosure which is described in more detail in numbered Examples 1-8.
  • the compounds 1-10 from Scheme A were used and/or synthesized in a glove box in a positive pressure argon atmosphere.
  • the solid support is controlled pore glass (CPG), where CPG is an exemplary solid support of the present disclosure.
  • Controlled pore glass (CPG) optionally, and typically does, include one or more of a plurality of reactive functional groups which may be reacted with a linking group precursor (e.g., HODA, (1) as shown in Scheme A) to provide compound (2).
  • the compound (2) is then coupled, or in other words linked, to a precursor of the compounds of the present disclosure, in this case through the hydroxyl group of a pentose ring of (3), to provide compound (4).
  • exemplary solid support SS namely CPG
  • Other reactive solid supports suitable for use in the present disclosure are known to the skilled person, and many of them are commercially available.
  • HQDA-CPG (2) was prepared according to the method of Pon et. al., "Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents," Bioconjugate
  • the supernatant was added to the CPG in the fritted syringe.
  • the syringe was capped on both ends and mixed on an inverting rotator for 2 hours.
  • the syringe was mounted on a vacuum manifold equipped with a stopcock, drained and sequentially washed with acetonitrile (3x5 mL), methanol (2x5 mL), acetonitrile (2x5 mL) and methylene chloride (2x5 mL) to provide the title compound (2) in pure form.
  • Confirmation of the HQ.DA coupling on the CPG was based on step trityl cation color formation as described in the following Example 2.
  • HBTU 121 mg, 319 ⁇
  • DMAP 39 mg, 319 ⁇
  • 5'-0-dimethoxytriyl-5-(octa-l,7-diynyl)-2'-deoxyuridine (3) 216 mg, 319 ⁇
  • acetonitrile 4.8 mL
  • This solution was added to dry HQDA-CPG (2) (1 g) in a separate polypropylene tube.
  • the tube was capped and mixed on an inverting rotator for 20 hours at room temperature.
  • the slurry was transferred to a syringe equipped with a frit and stopcock.
  • the syringe was mounted on a vacuum manifold and the reaction flow and acetonitrile wash (2 x 10 mL) were collected for subsequent recovery of uncoupled nucleoside.
  • the solid support was washed on the manifold with methanol (2 x 10 mL), acetonitrile (2 x 10 mL) methylene chloride (2 x 10 mL) and dried with vacuum.
  • the syringe was fitted with a closed stopcock and exposed to a solution of 16% 1-methylimidazole in THF (Pac 2 0 Cap B, 5 mL) followed by a solution of 5% (w/v) phenoxyacetic anhydride: 10% pyridine in THF (Pac 2 0 Cap A, 5 mL) was added to the dry CPG.
  • the syringe barrel was plugged with a plunger and mixed on an inverting rotator for 30 minutes.
  • the syringe was mounted on a vacuum manifold and washed with acetonitrile (3 x 10 mL), methylene chloride (2 x 10 mL) and dried with vacuum.
  • the solid support was deblocked by flowing 3% dichloroacetic acid in methylene chloride (8 x 10 mL). The solid support was washed with acetonitrile until colorless and then washed with acetonitrile (3 x 10 mL) and methylene chloride (2 x 10 mL). After the washes, the solid support was dried on the vacuum manifold to provide (4).
  • lodination of compound (4) was performed according to the method of Miller and Kool, "A Simple Method for Electrophilic Functionalization of DNA," Org. Lett., 4(21),3599-3601(2002).
  • Solid support bound 5-(octa-l,7-diynyl)-2'-deoxyuridine- 3'-0-HQ.DA-CPG (4) (330 mg) was transferred to a syringe fitted with a stopcock and loaded onto a vacuum manifold. The solid support was wetted with DM F (1 x 10 mL).
  • the solid support was rinsed with DMF, centrifuged, and the DMF supernatant was decanted.
  • the solid support was slurried with fresh DMF and transferred to a fritted syringe.
  • the solid support was washed with DMF (3 x 10 mL), acetonitrile (3 X 10 mL) and methylene chloride (2 x 10 mL).
  • the solid support was dried to a free flowing powder (6) on the vacuum manifold. See, e.g., Miller and Kool,”Versatile 5'-Functionalization on Solid Support: Amines, Azides, Thiols and Thioethers via Phosphorus Chemistry," J. Organic Chemistry, 69(7), 2404-2410 (2004).
  • a MS sample was prepared by transferring a small amount of (6) to a 2 mL screw cap tube. Cold NH 4 OH (500 ⁇ ) was added to the solid support and then incubated at room temperature for 5 minutes. The CPG/NH 4 OH slurry was transferred to a 3 mL syringe fitted with a 13 mm syringe filter with a 0.45 ⁇ GHP Acrodisc filter (Pall Corporation, Ft. Washington, NY). The plunger was fitted into the syringe barrel and the filtrate was collected into a 1.5 ml polypropylene tube.
  • the CPG/Acrodisc were washed with cold NH 4 OH (500 ⁇ ) followed by H 2 0 (500 ⁇ ) and added to the original filtrate. This solution was evaporated in a Savant Speedvac at 65°C for 1 hour followed by evaporation at room temperature to reduce the volume to at least 150 ⁇ .
  • the peak containing the 5'-azide (6) was sent to Numega Resonance Labs (San Diego, CA) for ESI MS analysis. The found m/z was in agreement with the calculated m/z for the structure shown below.
  • Phosphite (7) was prepared by dissolving 5-hexyn-l-yl-(2-cyanoethyl)- (N,N-diisopropyl)-phosphoramidite (403 ⁇ ) in dry acetonitrile (4 mL) with 0.28 M 5-ethylthio-lH-tetrazole (1.726 mL, 84 mmol). Methanol (28 ⁇ , 119 mmol) was added and sharp needles of ⁇ , ⁇ -diisopropylammonium ethylthiotetrazolide formed immediately. The solution was incubated at room temperature for 2 hours. The solution was separated from the crystals and the supernatant was divided into 4 polypropylene tubes and evaporated to form a solid mass (7).
  • the solid support was transferred to a fritted syringe, mounted on a vacuum manifold and washed with DMF (3 x 1 mL), acetonitrile (2 x 1 mL), H 2 0 (l x l mL) and acetonitrile (3 x 1 mL).
  • the solid (8) was dried by vacuum on the manifold.
  • a MS sample was prepared by transferring a small amount of (8) to a 2 mL screw cap tube. Cold NH 4 OH (500 ⁇ ) was added to the solid support and then incubated at room temperature for 5 minutes. The CPG/NH 4 OH slurry was transferred to a 3 mL syringe fitted with a 13 mm syringe filter with a 0.45 ⁇ GHP Acrodisc filter (Pall Corporation, Ft. Washington, NY). The plunger was fitted into the syringe barrel and the filtrate was collected into a 1.5 ml polypropylene tube.
  • the CPG/Acrodisc were washed with cold NH 4 OH (500 ⁇ ) followed by H 2 0 (500 ⁇ ) and added to the original filtrate. This solution was evaporated in a Savant Speedvac at 65°C for 1 hour followed by evaporation at room temperature to reduce the volume to at least 150 ⁇ .
  • the peak containing the (8) was sent to Numega Resonance Labs (San Diego, CA) for ESI MS analysis. The found m/z was in agreement with the calculated m/z for the structure shown below.
  • the diphosphate (9) and triphosphate (10) were prepared from 5'-N-(2- Cyanoethyl)-(5-hexyn-l-yl)-phosphoramidate-5-(octa-l,7-diynyl)-2',5'-dideoxyuridine- 3'-0-HQDA-CPG (8) (125 mg) on solid support using a MerMade 12 synthesizer.
  • the synthesis was performed with the following 2 basic automated steps: (R) Removal of the cyanoethyl phosphate protecting group; (C) Coupling of the Bis-CNET and oxidation to P(V) phosphate.
  • the sequence of additions and delivery volumes for these routines are set forth below:
  • the plunger was fitted into the syringe barrel and the filtrate was collected into a 1.5 ml polypropylene tube.
  • the CPG/Acrodisc were washed with cold N H 4 OH (500 ⁇ ) followed by H 2 0 (500 ⁇ ) and added to the original filtrate. This solution was evaporated in a Savant Speedvac at 65°C for 1 hour followed by evaporation at room temperature to reduce the volume to at least 150 ⁇ .
  • the crude material (11) was quantified by UV.
  • HPLC purification was performed on a Cadenza CD-C18 column (4.6mm x 150mm 3 ⁇ ) on the Prostar System using a gradient of 5 %B to 39.5 %B in 46 minutes at 1 mL/min and monitoring at 292 nm.
  • the peak containing the triphosphoramidate (11) was sent to Numega Resonance Labs (San Diego, CA) for ESI MS analysis.
  • Example 2 5'-0-dimethoxytriyl-5-(octa-l,7-diynyl)-2'-deoxyuridine (3) was employed as a starting material, so that the subsequently formed compounds 4- 11 each contained the uracil nucleobase.
  • This same synthetic route may be employed with suitable alternative octadiynyl 2'-deoxynucleosides to (3) so as to incorporate alternative nucleobases into a compound of Formula 1 as disclosed herein.
  • N6-protected 5'-DMT-2'-deoxy-7-octadiynyl-7-deazaadenosine N4- protected-5-octadiynyl-5'-DMT-2'-deoxycytidine, and N2-protected-5'-DMT-2'-deoxy- 7-octadiynyl-7-deazaguanosine may be used in lieu of the 5-octadiynyl-5'-DMT-2'- deoxyuridine (3).
  • Synthetic Scheme B provides an outline of a methodology according to the present disclosure which is described in more detail in numbered Examples 9-12.
  • the compounds 6 and 11-16 from Scheme B were used and/or synthesized in a glove box in a positive pressure argon atmosphere.
  • SAUCYL-(5-HEXYN-1-YL)-PHOSPHITE (14) [000137] In a polypropylene tube, 5-hexyn-l-ol (13) (25 ⁇ , 220 ⁇ ) was added to tributylamine (95 ⁇ , 400 ⁇ ) in DMF (343 ⁇ ). In a separate polypropylene tube, 2-chloro-4H-l,3,2-benzodioxaphosphorin-4-one (12) (81 mg, 400 ⁇ ) was dissolved in DMF (200 ⁇ _).
  • LNA Locked Nucleic Acids
  • R 1 is selected from
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 5 is selected from H and G 1 ;
  • R 6 is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13 is selected from
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and
  • R 12 form a direct bond
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom;
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • each of R 1 and R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen.
  • each of R 1 and R 13 is selected from an alkyl group and an oxyalkyi group, either of which terminates in a linker group (LG1), the LG1 bonded to a tether (T).
  • n R 6 The compound of embod n R 6 is selected from
  • R is selected from a) an alkyl group and an oxyalkyi group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and
  • G 1 is H or a protecting group
  • R 6 is a heterocycle comprising a substituent R 13 ;
  • R 8 is selected from OR 11 and O-L-SS where SS represents a solid support and resents a linking group between 0 and the SS;
  • R 11 is selected from H and G 3 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon ator 10)
  • the compound of embodiment 1 having the formula
  • R 6 is a heterocycle comprising a substituent R 13 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support bound to a linker R 11 is selected from H and G 3 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom 11)
  • the compound of embodiment 1 having a formula selected from the grou
  • R° is -OR or -O-L-SS where L-SS represents a solid support bound to a linker; R 11 is selected from H and G 3 ; and
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl; R 4 is selected from
  • R 6 is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13 is selected from
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R is -OR or -O-L-SS where L-SS represents a solid support bound to a linker
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and
  • R 12 form a direct bond
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom. 14) A process for forming a phosphate protected N-phosphoroamidate- monoester disphosphate (120) from a phosphoroamidate diester compound (110) and a phos horylating phosphoramidite compound 115,
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R 6 is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13 is selected from
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker (L);
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ; and G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;
  • R 2 is selected from hydrogen and Ci-C 4 alkyl
  • R is selected from R R and
  • R 6 is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13 is selected from
  • an alkyl group and an oxyalkyl group either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen; and b) an alkyl group and an oxyalkyl group, either of which terminates in a linker group (LG2), the LG2 bonded to the tether (T);
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker (L);
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 1 is H or a protecting group for a hydroxyl group that is bonded to a phosphorous atom
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom. 16) A process for forming a N-phosphoroamidate-monoester triphosphate (160) from a cyclotriphosphite (155) and an azide (105)
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon- carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and haloge
  • R 4 is selected from R 6 is a heterocycle, the heterocycle optionally comprising a substituent R 13 , where R 13 is selected from
  • R 7 is selected from hydrogen, -CH 2 -halogen, Ci-C 4 alkyl, hydroxyl and -CH 2 -OR 10 ;
  • R 8 is -OR 11 or -O-L-SS where L-SS represents a solid support optionally bound to a linker;
  • R 9 is hydrogen or, when R 7 is -CH 2 -OR 10 then R 9 may be -CH 2 -R 12 where R 10 and R 12 form a direct bond;
  • R 11 is selected from H and G 3 ;
  • G 2 is selected from oxygen, sulfur and CH 2 ;
  • G 3 is a protecting group for a hydroxyl group that is bonded to a carbon atom.
  • R 1 is selected from an alkyl group and an oxyalkyl group, either of which terminates in a functional group selected from carbon-carbon double bond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen.
  • any concentration range, percentage range, ratio range, or integer range provided herein is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term "about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated.

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EP15805684.6A 2014-11-20 2015-11-20 Nukleosidphosphoramidatester und derivate davon, verwendung und synthese davon Pending EP3221327A1 (de)

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